Int. J. Biol. Sci. 2016, Vol. 12 109

Ivyspring International Publisher International Journal of Biological Sciences 2016; 12(1): 109-119. doi: 10.7150/ijbs.12358 Research Paper The Complete Mitochondrial Genome of two Tetrag- natha (Araneae: Tetragnathidae): Severe Trun- cation of tRNAs and Novel Gene Rearrangements in Araneae Zheng-Liang Wang #, Chao Li #, Wen-Yuan Fang and Xiao-Ping Yu 

Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, Jiliang University, Hangzhou, Zhejiang, 310018, People’s Republic of China

# These authors contributed equally to this paper.

 Corresponding author: X.-P. Yu, Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang, People’s Republic of China. Phone and fax: 86-571-86836006. E-mail: [email protected].

© Ivyspring International Publisher. Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. See http://ivyspring.com/terms for terms and conditions.

Received: 2015.04.09; Accepted: 2015.10.26; Published: 2016.01.01

Abstract Mitogenomes can provide information for phylogenetic analysis and evolutionary biology. The Araneae is one of the largest orders of Arachnida with great economic importance. In order to develop mitogenome data for this significant group, we determined the complete mitogenomes of two long jawed spiders maxillosa and T. nitens and performed the comparative analysis with previously published mitogenomes. The circular mitogenomes are 14578 bp long with A+T content of 74.5% in T. maxillosa and 14639 bp long with A+T content of 74.3% in T. nitens, respectively. Both the mitogenomes contain a standard set of 37 genes and an A+T-rich region with the same gene orientation as the other spider mitogenomes, with the exception of the dif- ferent gene order by the rearrangement of two tRNAs (trnW and trnG). Most of the tRNAs lose TΨC arm stems and have unpaired amino acid acceptor arms. As interesting features, both trnSAGN and trnSUCN lack the dihydrouracil (DHU) arm and long tandem repeat units are presented in the A+T-rich region of both the spider mitogenomes. The phylogenetic relationships of 23 spider mitogenomes based on the concatenated nucleotides sequences of 13 protein-coding genes in- dicated that the mitogenome sequences could be useful in resolving higher-level relationship of Araneae. The molecular information acquired from the results of this study should be very useful for future researches on mitogenomic evolution and genetic diversities in spiders.

Key words: Mitogenome; Araneae; Tetragnatha maxillosa; Tetragnatha nitens; Phylogeny

Introduction Generally, the mitogenome is a circu- nome size, maternal inheritance, high rate of evolu- lar, double-stranded molecule, ranging in size from 14 tion and low level of intermolecular genetic recom- kb to 20 kb, usually containing a standard set of 13 bination, the mitogenome has been widely regarded protein-coding genes (PCGs), two ribosomal RNA as an effective molecular marker for molecular evolu- genes (the large and small ribosomal subunits, tion, phylogenetic studies, population genetics and rRNAs), 22 transfer RNA genes (tRNAs) and an phylogeography [2]. Moreover, the information of A+T-rich region in a highly variable length with es- gene rearrangement, tRNA secondary structure, ge- sential regulatory elements for replication and tran- netic code alteration and models of control of replica- scription [1]. Due to the characteristics of small ge- tion and transcription in the complete mitogenome

http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 110 data are also extensively exploited for deep-level Materials and methods phylogenetic inference at various taxonomic levels in the past decade [3,4]. Sample collection and DNA extraction Spiders (Araneae) are among the largest Specimens of spiders, T. maxillosa and T. nitens groups in the world due to their broad diversity, were collected from the paddy fields in Yuyao world-wide distribution and conspicuous synapo- (E121°09′, N30°03′), Zhejiang Province, China. The morphies [5]. Approximately 45,000 described field collections did not involve endangered or pro- in 114 families make Araneae the seventh-largest or- tected species and no specific permits were required der next to the five largest insect orders (Coleoptera, for our collecting. All collections were preserved in , , Diptera and ) 95% ethanol and stored in the temperature cabinet at and Acari among the Arachnida [6]. Due to such high 4°C. Total genomic DNA was extracted from leg tis- species diversity, there are many taxonomic problems sue of each spider individual of the two Tetragnatha about the order Araneae which remain to be unsolved species using the DNeasy Tissue Kit (Qiagen, Hilden, [7,8]. Because of homoplasies or reduction of ana- Germany) according to the manufactures protocol. tomical characters in morphological and ethological data [5], it would be desirable to examine an inde- PCR amplification, cloning and sequencing pendent set of molecular data. Partial mitochondrial Primer sequences of long PCR amplification gene sequences of cytochrome oxidase subunit 1 were designed based on the conserved sequences (COI), rRNAs (16S and 12S) and tRNAs, and nuclear which were obtained by aligning the complete mito- genes of rDNA (18S and 28S) and histone (H3) from genome sequences of four spider species that were spiders have been documented and extensively used downloaded from GenBank: oregonnesis for phylogenetic analysis [9,10]. However, these short (NC_005942), Heptathela hangzhouensis (NC_005924), mitochondrial or nuclear genes usually lack the phy- Nephila clavata (NC_008063) and Telamonia vlijmi logenetic information in resolving phylogenetic rela- (NC_024287). The six primer sets that were used to tionships among families or subfamilies within one amplify overlapping fragments of the complete mi- order and sometimes resulted in controversial signal togenome of T. maxillosa and T. nitens are shown in [8,11]. Hence, additional reliable data sets for phylo- Table S1. genetic reconstructions, such as the complete mito- All PCRs were performed using TaKaRa LA PCR genome sequences, are required. Amplification Kit Version 2.1 (Takara Co., Dalian, To date, more than 130 complete mitogenomes China) with the following amplification condition: an from species of have been determined, but initial denaturation for 5 min at 95°C, followed by 40 only 21 species in 14 families from Araneae were cycles of denaturation for 10s at 92°C, annealing for publicly available in GenBank. These 21 mitogenomes 30s at 50°C, elongation for 4 min at 68°C in the initial share a similar molecular size, ranging from 13,874 bp 20 cycles and then increase by 20s per cycle in the final in Ornithoctonus huwena [12] to 14,741 bp in Wadicosa 20 cycles and a final extension step for 10 min at 72°C. fidelis [13], but have variations in mitogenomic char- All PCR products were resolved by electrophoresis in acteristics, such as gene content and gene arrange- 1.0% agarose gel, purified with DNA Gel Purification ment. mitogenomes, in particular spider Kit (TianGen, China), ligated to the pGEM-T-Easy mitogenomes, possess compact mitogenomes and vector (Promega,USA) and sequenced using DNA their tRNAs usually lack the sequence for the T-arm Sequencer (ABI377) at Sangon Inc (Shanghai, China). and a paired aminoacyl stem of the cloverleaf struc- ture. Such atypical tRNAs might be modified by Sequencing assembling and annotation post-transcriptional editing to be functional [14]. The complete mitogenome sequences were as- In the present study, we sequenced and anno- sembled using the Staden package 1.7.0 [15]. PCGs tated the complete mitogenome sequences of two long and rRNAs were initially identified via the MITOS jawed spiders Tetragnatha maxillosa and T. nitens web server [16] and subsequently refined by hand (Araneae: Tetragnathidae), which are the dominant annotation method following the procedures pro- arthropod predators in the agroecological system. 21 posed by Cameron [17]. Gene boundaries were de- spider mitogenomes that are available in GenBank termined by comparing with gene regions from pre- were used in the comparative analysis to reveal the viously characterized spider mitogenomes: W. fidelis gene rearrangements in two Tetragnatha species. Ad- [13], O. huwena and H. hangzhouensis [12]. tRNAs were ditionally, phylogenetic study was also performed to identified by tRNAscan-SE 1.2.1 [18] and ARWEN assess the utility of mitogenome data to understand 1.2.3.c [19]. The putative tRNAs which were not found higher phylogeny of Araneae. by the two software tools were identified based on sequence similarity to tRNAs of the other previously

http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 111 published spider mitogenomes. Nucleotide composi- other spider mitogenomes available from GenBank tion and codon usage were calculated in MEGA 4.0 [13]. Both mitogenomes share the same 37 typical [20]. Potential inverted repeats or palindromes in the metazoan gene set (13 PCGs, 22 tRNAs and two A+T-rich region were determined using Tandem Re- rRNAs) and an A+T-rich region [1]. Among these 37 peats Finder (http://tandem.bu.edu/trf/trf.html) genes, twenty-three are coded on the major strand with the default parameters [21]. (J-strand) while the rest are coded on the minor strand AT-skew=(A–T)/(A+T) and GC-skew=(G–C)/(G+C) (N-strand). Gene orientation of T. maxillosa and T. were used to measure the nucleotide compositional nitens mitogenomes is identical to that of all previ- difference between genes [22]. ously determined araneoid mitogenomes [27,28]. The complete mitogenome sequences of T. max- With the exception of eight translocated tRNAs, mi- illosa and T. nitens have been deposited in the Gen- togenome gene order of the two species are similar to Bank database under accession number KP306789 and that of L. polyphemus, which is considered to represent KP306790, respectively. the putative ground pattern of the [23]. As in most of the arthropod mitogenomes, the Phylogenetic analysis nucleotide composition of the J-strands shows a A total 21 spider mitogenomes available in the highly A+T content which accounts for 74.5% in T. GenBank were included in the phylogenetic analysis maxillosa and 74.3% in T. nitens, respectively. Meta- (Table 1). Limulus polyphemus (Xiphosura) was se- zoan mitogenomes usually present a clear strand bias lected as an outgroup [23]. To overcome of base in nucleotide composition [29], and arthropod mito- compositional heterogeneity and explore the effect of genomes tend to have a positive AT-skew and a neg- method choice on phylogenetic reconstruction, three ative GC-skew on the J-strand [30]. However, the datasets were assembled: (1) a concatenated nucleo- strength of the skew in both T. maxillosa and T. nitens tide sequence alignment of 13 PCGs (PCG123); (2) a mitogenomes shows the opposite pattern, as the concatenated nucleotide sequence alignment of the AT-skew and GC-skew in the J-strands are –0.087 and first and second codons of 13 PCGs (PCG12); (2) a 0.246 in T. maxillosa, –0.055 and 0.200 in T. nitens, re- concatenated nucleotide sequence alignment of the spectively (Table 1). The similar patterns of nucleotide first and second codons of 13 PCGs and two rRNAs skew are also commonly found in mitogenomes of (PCR12R). The gene order of 13 PCGs in three con- other taxa, such as in those of scorpions catenated datasets is as follows: ATP6, ATP8, COI, [31]. Through comparison of the nucleotide skews COII, COIII, Cytb, ND1, ND2, ND3, ND4, ND4L ND5 among all the sequenced spider mitogenomes, the and ND6. Both Maximum Likelihood (ML) and divergence between the two suborders was detected. Bayesian Inference (BI) were used to infer phyloge- All the Opisthothelae spider mitogenomes exhibit a netic trees. The GTR+I+G was selected as the best negative AT-skew but a positive GC-skew, while mi- model for three nucleotide datasets under the Akaike togenomes of two species from Meaothelae are char- information criterion by ModelTest 3.7 [24]. ML acterized by a positive AT-skew and negative analysis was performed by a PHYML online web GC-skew. Similar phenomenon was also observed in server [25] with default parameters and the node pseudoscorpion mitogenomes [32]. These results in- support values were evaluated via a bootstrap test dicate that their ancestors underwent a reversal bias with 100 replicates. BI analysis was conducted by in nucleotide composition on the J-strand between the MrBayes version 3.1.2 [26]. Two set of four chains (one two suborders. The nucleotide composition and cold chain and 3 hot chains) were allowed to run skewness in different gene regions of T. maxillosa and simultaneously for 1,000,000 generations and trees T. nitens mitogenomes are shown in Table S2. were sampled every 1,000 generations, with the first In the T. maxillosa mitogenome, there are 10 in- 25% discarded as burn-in. Stationarity was considered tergenic spacers with a total of 135 bp long varying to be reached when the average standard deviation of from 1 bp to 41 bp. The three long intergenic spacers split frequencies was less than 0.01. are located between trnN and trnA (41 bp), COIII and ND3 (35 bp), and trnG and trnM (30 bp), respectively. Results and Discussions The T. nitens mitogenome has 12 intergenic spacers Genome organization, structure and compo- with a total of 139 bp with various lengths of 2-38 bp. sition The longest intergenic spacer is situated between COIII and ND3. Gene overlaps in the mitogenome of The complete mitogenome sequences of T. max- T. maxillosa are found in 20 locations and their total illosa and T. nitens are 14,578 bp and 14,639 bp in size, size is 166 bp. Size of each overlapped region varies respectively (Fig. 1). The relatively small mitogenome from 2 to 22 bp. 17 overlapped regions are found in sizes (<15 kb) are well within the observed range of the mitogenome of T. nitens and their total size is 143

http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 112 bp. Size of each overlapped region ranges from 1 to 23 in T. maxillosa and T. nitens mitogenomes, respective- bp. Overlapping tRNAs have long been known ly, and the longest overlap occurs between trnE and throughout metazoans [33]. There are 7 and 8 over- trnF in both of the spider mitogenomes. laps which are located between two adjacent tRNAs

Fig. 1. Map of mitogenome of T. maxillosa (A) and T. nitens (B). Transfer RNAs are designated by the IUPAC-IUB single letter amino acid codes (L1: trnLCUN; L2: trnLUUR; S1: trnLAGN; L1: trnLUCN) and those encoded by major- and minor-strand are shown outside and inside of the circular mitogenome map, respectively. Gene names not underlined indicate a clockwise direction of transcription, and those with underline indicate a counterclockwise direction transcription.

Table 1. Nucleotide composition bias of all spider mitogenomes available from GenBank and an outgroup species used in phylogenetic analysis.

Order Family Species Length (bp) AT-skew GC-skew Accession number Araneae Tetragnathidae Tetragnatha maxillosa 14578 –0.087 0.246 KP306789 Tetragnathidae Tetragnatha nitens 14639 –0.055 0.200 KP306790 Araneidae amoena 14121 -0.060 0.241 NC_024281* Araneidae Argiope bruennichi 14063 -0.035 0.263 NC_010596* Araneidae Araneus ventricosus 14617 -0.048 0.243 NC_025634* Araneidae Neoscona theisi 14156 -0.059 0.252 NC_026290* Salticidae Habronattus oregonensis 14381 -0.112 0.301 NC_005942* Salticidae Telamonia vlijmi 14601 -0.081 0.235 NC_024287* Salticidae paykull 14316 -0.105 0.318 NC_024877* Lycosidae Pirata subpiraticus 14528 -0.111 0.295 NC_025523* Lycosidae Pardosa laura 14513 -0.109 0.285 NC_025223* Lycosidae Wadicosa fidelis 14741 -0.130 0.312 NC_026123* Pholcus sp. 14279 -0.188 0.372 NC_010775* Pholcidae Pholcus phalangioides 14459 -0.191 0.371 NC_020324* Phyxioschema suthepium 13931 -0.040 0.472 NC_020322* Hypochilidae thorelli 13991 -0.140 0.266 NC_010777* Calisoga longitarsis 14070 -0.146 0.365 NC_010780* Nephilidae Nephila clavata 14436 -0.053 0.242 NC_008063* Theraphosidae Ornithoctonus huwena 13874 -0.083 0.344 NC_005925* Selenops bursarius 14272 -0.123 0.321 NC_024878* Oxyopidae Oxyopes sertatus 14442 -0.130 0.321 NC_025224* Liphistius erawan 14197 0.024 -0.361 NC_020323* Heptathelidae Heptathela hangzhouensis 14215 -0.023 -0.235 NC_005924* Xiphosura Limulidae Limulus polyphemus 14985 0.114 -0.399 NC_003057* * refers to the mitogenomes that have been downloaded from GenBank.

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Protein-coding genes and codon usage two spider mitogenomes with the exception of TAA as stop codon for COII and ND1 in T. nitens. The in- Among 13 mitochondrial PCGs of the two complete termination codon was commonly found in Tetragnatha species, only four genes (ND5, ND4, ND4L metazoan mitogenomes, which is presumed to be and ND1) are coded on the N-strand, while the other completed via post-transcriptional polyadenylation genes are all coded on the J-strand (Table 2). Start [34]. In accordance with other arthropod mitoge- codons follow the ATN rule in all 13 PCGs, except of nomes, overlapping regions between two adjacent three genes (COI, COII and COIII). The start codon for PCGs are present in both T. maxillosa and T. nitens COI is TTT in T. maxillosa and TTA in T. nitens, re- mitogenomes [35]. A 4 bp overlap exists in the junc- spectively. Both COII and COIII use TTG as an initial tion between ATP8 and ATP6 in both spider mitoge- codon in T. maxillosa and T. nitens. The start codons of nomes. Generally, hairpin structures forming at the 3' TTT or TTA for COI and TTG for COII and COIII are end of the upstream protein's mRNA may act as a also presented in mitogenomes of many species, es- signal for the cleavage of the polycistronic primary pecially for spiders [12,13]. The standard stop codons transcript [36]. Either incomplete stop codons or (TAA and TAG) and one incomplete stop codon (T) overlaps between genes may be a product of the se- are utilized in all 13 PCGs. In T. maxillosa, three genes lective pressure to reduce genome size noted in mi- (COII, Cytb and ND1) terminate with TAG, two genes tochondria [37]. (ND2 and ND4L) use T and the rest genes are all stop with TAA. The stop codons are identical between the

Table 2. Mitochondrial genome organization of Tetragnatha maxillosa and T. nitens*.

Feature Strand T. maxillosa T. nitens Position Spacer (+)/Overlap (–) Start/Stop codon Position Spacer (+)/Overlap (–) Start/Stop codon tRNAMet J 1–62 +30 1–61 0 ND2 J 53–986 –10 ATT/T 48–981 –14 ATT/T tRNATyr N 989–1043 +2 985–1040 +3 tRNACys N 1032–1094 –12 1030–1095 –11 COI J 1093–2628 -2 TTT/TAA 1092–2627 –4 TTA/TAA COII J 2632–3291 +3 TTG/TAG 2631–3290 +3 TTG/TAA tRNALys J 3288–3342 –4 3293–3344 +2 tRNAAsp J 3334–3393 –9 3338–3398 –7 ATP8 J 3385–3540 –9 ATT/TAA 3399–3542 0 ATA/TAA ATP6 J 3537–4199 –4 ATA/TAA 3539–4201 –4 ATA/TAA COIII J 4203–4988 +3 TTG/TAA 4205–4990 +3 TTG/TAA ND3 J 5024–5344 +35 ATA/TAA 5029–5343 +38 ATT/TAA tRNALeu(UUR) N 5331–5385 –14 5332–5387 –12 tRNAAsn J 5381–5439 –5 5385–5441 –3 tRNAAla J 5481–5546 +41 5474–5535 +32 tRNASer(AGN) J 5544–5598 –3 5534–5584 –2 tRNAArg J 5600–5650 +1 5581–5633 –4 tRNAGlu J 5643–5700 –8 5624–5682 –10 tRNAPhe N 5679–5732 –22 5660–5714 –23 ND5 N 5734–7377 +1 ATC/TAA 5713–7356 –2 ATA/TAA tRNAHis N 7372–7424 –6 7354–7405 –3 ND4 N 7425–8708 0 ATA/TAA 7426–8712 +20 TTG/TAA ND4L N 8717–8976 +8 ATT/T 8713–8977 0 ATT/T tRNAPro N 8963–9020 –14 8965–9024 –13 ND6 J 9033–9461 +12 ATA/TAA 9034–9462 +9 ATA/TAA tRNAIle J 9459–9518 –3 9461–9513 –2 Cytb J 9510–10643 –9 ATA/TAG 9516–10643 +2 ATT/TAG tRNASer(UCN) J 10639–10694 –5 10652–10705 +8 tRNAThr J 10695–10752 0 10706–10761 0 ND1 N 10742–11695 –11 ATT/TAG 10743–11684 –19 ATA/TAA tRNALeu(CUN) N 11690–11746 –6 11693–11762 +8 rrnL N 11747–12769 0 11763–12769 0 tRNAVal N 12770–12829 0 12770–12826 0 rrnS N 12830–13517 0 12827–13516 0 tRNAGln N 13518–13574 0 13517–13576 0 tRNATrp J – – – – 13588–13641 +11 tRNAGly J – – – – 13632–13695 –10 CR J 13575–14438 0 13696–14639 0 tRNATrp J 14439–14492 0 – – – – tRNAGly J 14484–14548 –9 – – – – * J and N refer to the major and minor strand, respectively. Position numbers refer to positions on the major strand.

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The A+T contents of all the 13 mitochondrial are commonly observed in many metazoan mitoge- PCGs, excluding stop codons, are 73.5% in T. maxillosa nomes including the sea spider Achelia bituberculata and 73.0% in T. nitens, respectively. The third codon [40], the H. oregonensis [41], and the position has a relatively higher A+T content (83.9% in camel spider Nothopuga sp. [42]. However, missing of T. maxillosa and 83.1% in T. nitens) than the first (68.7% the DHU-arm in trnSUCN is not a common feature ob- in T. maxillosa and 68.5% in T. nitens) and second served in arthropod mitogenomes, though it has been (67.9% in T. maxillosa and 67.3% in T. nitens) codon found in some chelicerate mitogenomes [13]. Addi- positions. In addition, both the second and third co- tionally, lots of unmatched base pairs and G–U wob- don positions in T. nitens and all of the three codon ble pairs scatter throughout all of tRNA secondary positions in T. maxillosa have negative AT-skews. structures in both spider mitogenomes and most of These AT-bias might affect codon usage in proteins, the unmatched base pairs occur on the amino acid with ATT (encoding Ile) and TTT (encoding Phe) be- acceptor arm. Such aberrant secondary structures are ing the most frequently used codons in mitogenomes also found in the spiders H. oregonensis [41], Heptathela of T. maxillosa and T. nitens, respectively. The relative hangzhouensis and O. huwena [12]. To be functional, synonymous codon usage (RSCU) in 13 PCGs of the these noncanonical tRNAs may require coevolved two spider mitogenomes is summarized in Fig. S1. interacting factors or post-transcriptional RNA edit- The usage of both two-fold and four-fold degenerate ing [41,43]. For examples, seryl-tRNA synthetase has codon is biased to use the codons which are abundant evolved to be able to recognize the noncanonical in A or T in the third position, and GC-rich codons are tRNA-Ser in mammalian mitochondria [43] and a likely to be abandoned. These features are consistent template-dependent RNA editing mechanism has with other arthropod mitogenomes [30]. been demonstrated in centipede Lithobius forficatus: the 3′ end sequences of the acceptor stem act as a Transfer and ribosomal RNA genes template to synthesize the matching strand [14]. Since The complete set of 22 tRNA genes typically organisms that possessed the similar non-canonial found in metazoan mitogenomes is present in the two tRNA structures might be share the evolutionary spider mitogenomes: two tRNAs for each of serine history and have a much closer lineage [44], the in- and leucine, and one tRNA for each of the other 18 formation of tRNA secondary structures could be amino acids. All tRNAs are scattered throughout the used for deep-level phylogenetic inference. Masta and circular mitogenome and varied in size from 51 bp Boore (2008) have reported that T-arm loss in trnR, (trnR) to 66 bp (trnA) in T. maxillosa and ranged from trnK and trnM occurred only once and could be a 51 bp (trnR) to 70 bp (trnI) in T. nitens. The concate- synapomorphy for Opisthothelae spiders [44]. In case nated sequence of all 22 tRNAs shows a high A+T of the two Tetragnatha mitogenomes, however, the biased, which accounts for 78.1% in T. maxillosa and typical cloverleaf structure could be predicted for 79.1% in T. nitens, and exhibits a negative AT-skew trnM gene, although the stem is short with only two (–0.012) in T. maxillosa and a positive GC-skew (0.188) complementary base pairs in T. nitens (Fig. 2). in T. nitens, respectively (Table S2). Among 22 tRNAs, The ends of rRNAs are difficult to be precisely 14 tRNAs are coded on the J-strand and the rest on the determined by DNA sequencing alone, so they are N-strand. assumed to extend to the boundaries of flanking The predicted secondary structures of 22 tRNAs genes. As in mitogenomes of H. oregonensis [41] and H. in two spider mitogenomes are shown in Fig. 2. Most hangzhouensis [12], the two rRNAs are located be- of them cannot be folded into typical clover- tween trnLCUN and trnV, and between trnV and trnQ, leaf-shaped secondary structures. Nine of the tRNA respectively. The length of rrnL is 1023 bp with an sequences lack the potential to form TΨC arm stem A+T content of 79% in T. maxillosa and 1007 bp with pairings. Instead, they are inferred to have an A+T content of 78.3% in T. nitens, respectively. The TV-replacement loops. These extremely truncated rrnS is 688 bp with an A+T content of 81.2% in T. tRNAs might be resultant products of selection pres- maxillosa and 690 bp with the A+T content of 82.5% in sure for minimizing the size of Araneae mitogenomes. T. nitens, respectively. In addition, both rRNAs exhibit The trnSAGN and trnSUCN in both spider mitogenomes a negative AT-skew and a positive GC-skew in T. lack the dihydrouracil (DHU) arm, but are simplified maxillosa. In T. nitens, however, only the rrnL shows a down to a loop. Although the trnSAGN show typical negative AT-skew and a positive GC -skew, whereas clover-leaf structure in the mitogenomes of some ex- rrnS exhibits a positive AT-skew and a negative ceptional species such as Adoxophyes honmai [38] and GC-skew. Pseudocellus pearsei [39], missing of the dihydrouracil (DHU) arm in the secondary structure of the trnSAGN

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Fig. 2. Inferred secondary structure of 22 tRNAs of T. maxillosa (Tm) and T. nitens (Tn) mitogenomes. The tRNAs are labeled with the abbreviations of their corresponding amino acids. Dashed (–) lines indicate Watson-Crick base pairing and centered dots (●) indicate G-U base pairing.

The A+T-rich region 690 bp and contains 71.5% A+T content with both positive AT-skew and GC-skew. The sizes of A+T-rich The A+T-rich region in mitogenome is also region in T. maxillosa and T. nitens are consistent with called control region (CR), and essential for the initia- those of the A+T-rich region of other spiders, which tion of replication in vertebrates [45,46]. The A+T-rich range from 387 bp in Phyxioschema suthepium to 1078 region in T. maxillosa is located between trnQ and bp in Pholcus phalangioide, but relatively shorter than trnW, spans 864 bp with 73.3% A+T content and those of most insect mitogenomes [30]. Moreover, two shows a negative AT-skew (-0.055). However, this spider species share 55.0% sequence identity in this region is flanked by trnG and trnM in T. nitens, spans region and both have a conserved T-stretch (28 bp) in

http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 116 the initial quarter of the A+T-rich region on the the super-families of Hypochilidae and Pholcoidea N-strand, which is thought to be responsible for the belonging to and the super-families control of replication and transcription of arthropod of Theraphosoidea, Nemesioidea and Dipluroidea mitogenomes [47]. belonging to formed a phylogenet- As the largest noncoding part of the metazoan ically closely related clade that was well supported by mitogenome, the A+T-rich region shows a higher size BI tree (0.97), in spite of the lower bootstrap support- variation than the other regions of the mitogenome ing value (52) in ML tree. Increased taxon sampling due to both length variation with tandem repeat units can significantly reduce phylogenetic error. Hence, (TRs) and differences in their copy numbers. There are additional mitogenomes data from the infraorders four long TRs present in both spider mitogenomes. Araneomorphae and Mygalomorphae are required to The length of the first three TRs are 172 bp in T. max- better establish the phylogeny of Opisthothelae. illosa and 180 bp in T. nitens, respectively, and the fourth TR is slightly shorter, which is 152 bp (1 bp Rearrangement of mitochondrial gene order overlapped with adjacent trnW) in T. maxillosa and 174 in Araneae bp (6 bp overlapped with downstream trnM) in T. When compared with the putative ancestral ar- nitens, respectively. The TR motifs of the two spiders thropod mitogenome as presented by L. polyphemus share 63.0% sequence identity and are rich in GC in [23], eight tRNAs are found to translocate their posi- the beginning of this region. The TRs could be dupli- tions in both Tetragnatha spider mitogenomes (Fig. 3). cated through slipped-strand mispairing and differ- The trnI changes its position to a new location be- ences in their numbers were considered as the source tween ND6 and Cytb. This tRNA rearrangement is of size variation in the entire mitogenome [30]. also found in most of Araneomorphae spiders, such as Stem-loop structures were also detected in the TR H. oregonensis [41], T. vlijmi [49] and N. clavata [27]. region of the two spider species. Two large hairpin Additionally, trnLCUN, trnN and trnSAGN rearrange structures could be potentially formed in the TR of T. their positions in both Tetragnatha spider mitoge- maxillosa, and similar complicated hairpin structures nomes, and these three tRNAs translocations are seem were also shown in the TR of T. nitens (Fig. S2). to be a conserved rearrangement event in all Opis- thothelae species. Tandem duplication random dele- Phylogenetic analysis tion events and transposition of single tRNAs are Phylogenetic analysis was performed on three considered to be the predominant mechanism of mi- concatenated nucleotide datasets from 23 Araneae togenome rearrangement [50]. When the total 21 spi- species and an outgroup species. The final alignment der mitogenomes that have been sequenced to date resulted in 11,241 sites for dataset PCG123, 7,494 sites were analyzed, the arthropod ground pattern is found for dataset PCG12 and 9,529 sites for dataset PCG12R, to be retained in two spiders (Liphistius respectively. The topologies of phylogenetic trees in- erawan and H. hangzhouensis). Six spider species (Cali- ferred from two methods (ML and BI) and three da- soga longitarsis, Hypochilus thorelli, O. huwena, P. phal- tasets were almost identical (Fig. 4, Fig. S3). The re- angioides, Pholcus sp. and P. suthepium) share the same sults clearly indicated that T. maxillosa and T. nitens six translocated tRNAs, and a subsequent trnI trans- share a close ancestry with N. clavata. Tetragnathidae location in nine spider species (Argiope bruennichi, H. as sister group of other araneoids (Nephilidae and oregonensis, T. vlijmi, A. amoena, Selenops bursarius, Araneidae) is quite well supported both by BI and ML Plexippus paykulli, Oxyopes sertatus, Pardosa laura and analysis, consistent with earlier studies that used Pirata subpiraticus) and trnM is rearranged in N. clav- morphological and molecular data [7]. Our findings ata. The new finding in present study is the novel gene also provide strong support for the monophyly of order rearrangement of trnG and trnW, which has not Araneoidea [8], which is placed as a sister relationship been observed in all above Araneae mitogenomes yet. to + Selenopoidea + Salticoidea The trnW and trnG are translocated to the new posi- (RTA-clade, named for the retrolateral tibal apophysis tion between the A+T-rich region and trnM in T. max- on the male copulatory organs). Moreover, the results illosa, and between trnQ and A+T-rich region in T. also strongly supported the monophyly of two sub- nitens, respectively. Whether this rearrangement orders (Opisthothelae and Mesothelae) in Araneae event is limited to only the spiders belonging to with high supporting values. However, the subdivi- Tetragnathidae? To answer this, further mitogenomes sion of Opisthothelae into two infraorders (Arane- sequencing of closely related taxa would be required. omorphae and Mygalomorphae) was not supported Moreover, our data suggest that the phylogenetic re- in the phylogenetic trees, although the monophyly of lationships among the Araneae can be also distin- the two infraorders has been broadly accepted by guished by gene order synapomorphies. We present a most of the arachnologists [48]. As illustrated in Fig. 4, parsimonious scenario of gene order changes and

http://www.ijbs.com Int. J. Biol. Sci. 2016, Vol. 12 117 map these rearrangement events on the phylogenetic spiders T.maxillosa and T. nitens were characterized trees inferred from the nucleotide dataset of PCG123. and compared with those of other Araneae species. As shown in Fig. 4, the taxa from Mesothelae in basal The complete mitogenomes of both Tetragnatha spi- clade have the same gene order as L. polyphemus, ders reveal a typical circular molecule with a similar suggesting that gene order rearrangements might gene content and orientation as found in other meta- occur after the divergence of the Araneae suborders. zoans. The comparative analysis suggests that the Translocation of four tRNAs (trnN, trnQ, trnS1 and gene orders of the Tetragnatha mitogenomes are trnL2) and inversion of two tRNAs (between trnY and moderately rearranged and represent new patterns. trnC) could be a synapomorphy for Opisthothelae With exceptions of the similar translocation events spiders. Likewise, reverse translocation of trnI in spe- shared by other sequenced spider mitogenomes, two cies of RTA-clade + Araneoidea and translocation of tRNAs (trnG and trnW) in the two Tetragnatha mito- trnI in species belong to Hypochiloidea, Dipluridae, genomes were found in different positions. Most of Theraphosidae, Nemesiidae and Plolicidae likely ex- the tRNAs lose TΨC arm stems and have perienced parallel evolution after Opisthothelae di- TV-replacement loops instead. Likely to the trnS (AGN), vergence. Additionally, translocation of trnM is the trnS(UCN) lacks the DHU arm in both spider mito- unique to a particular spider taxon (N. clavata) and genomes. Phylogenetic analysis indicates that mito- rearrangement of trnW and trnG seems to be a syn- genome sequences are useful in resolving higher-level apomorphy for the Tetragnatha. Overall, our relationship of Araneae. Data on genome arrange- results imply that the complicated gene rearrange- ment provide further information to help resolve re- ment in mitochondria could be considered as one of lationships among the spiders. The present results of key characters in inferring higher-level phylogenetic T. maxillosa and T. nitens mitogenomes will elevate our relationship of Araneae. knowledge on mitogenomes of Araneae. However, future research including additional taxon sampling Conclusion is needed to determine rearrangement mechanisms The complete mitogenomes of two long jawed and evolutionary processes.

Fig. 3. Schematic representation of mitochondrial gene arrangements in Araneae species compared to the putative ancestral arthropod gene order. Underlined protein coding genes and tRNAs are encoded in the minor strand. Genes that changed the positions were marked by numbers below, and the numbers refer to putative rearrangement events as follows: ①Translocation of trnL2; ②Translocation of trnN; ③Translocation of trnS1; ④Translocation of trnQ; ⑤Inversion between trnY and trnC; ⑥Reverse translocation of trnI; ⑦Translocation of trnI; ⑧Translocation of trnW; ⑨Translocation of trnG; ⑩Translocation of trnM.

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Fig. 4. Phylogenetic tree from Araneae species based on nucleotide sequence of 13 mitochondrial protein-coding genes using Bayesian inference (BI) and maximum likelihood (ML). Limulus polyphemus was used as an outgroup. Numbers above the nodes refer to Bayesian posterior probabilities in percentages (left) and ML bootstrap values (right). Hypothesized gene translocations for spiders are denoted on the phylogenetic tree by numbered circles as shown in Fig. 3.

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